The p53 network is the most commonly deregulated gene circuitry in human cancer. More than half of tumors carry mutations in the TP53 gene, and in the remaining fraction p53 is likely attenuated by hyperactivation of repressors such as MDM2. Activated p53 participates in various cellular responses to stress including cell cycle arrest and apoptosis. It is estimated that 11 million patients worldwide carry tumors with a normal version of the p53 protein, which could potentially be reactivated for selective elimination of cancer cells. Thus, the design of therapies exploiting the tumor suppressive function of p53 is a top priority in modern medicine. However, these efforts are hampered by the fact that p53 is a highly pleiotropic factor, in the sense that it delivers cell death only in some scenarios. What determines whether cells die or survive in response to p53 activation? p53 is a transcription factor that binds to DNA and activates the expression of hundreds of target genes, only some of which are involved in apoptosis. Depending on the context, some p53 target genes are expressed more strongly than others, and this variability affects cell fate. The goal of our research program is to elucidate the molecular mechanisms driving gene-specific regulation within the p53 network. In this grant proposal we report three novel molecular mechanisms mediating the selective regulation of p53 target genes. Each of these mechanisms will be further investigated by developing the following Specific Aims: 1. To define how CTCF, Polycomb complexes and lncRNAs regulate PUMA. We discovered that the potent apoptotic p53 target gene PUMA is regulated by a novel mechanism involving the insulator protein CTCF, the chromatin regulatory complexes PRC1-2 and two long non-coding RNAs. We will dissect the functional interplay between these factors at the PUMA locus using innovative experimental approaches. These efforts may enable strategies to selectively increase expression of this apoptotic gene in cancer cells. 2. To elucidate the mechanism of gene-specific repression by ??Np63?. We discovered that the oncoprotein ??Np63? shuts down a large subset of p53 target genes by a novel mechanism involving various repressive epigenetic events, such as deposition of histone variants and histone deacetylation /demethylation. We will investigate the role of various ??Np63? corepressors and ??Np63? protein domains in this novel mode of gene regulation, which may reveal strategies to target this potent oncogene for therapeutic purposes. 3. To characterize novel regulators of the p21: PUMA expression ratio identified by a genome-wide shRNA screen. We completed a genetic screen in cancer cells to identify factors regulating the balance between p21, the key mediator of p53-dependent cell cycle arrest, and PUMA. We will now investigate the mechanism of action of these coregulators, which could be targeted to define cell fate upon p53 activation. These studies will significantly advance our understanding of how p53 target genes are differentially regulated, and also contribute toward the design of strategies manipulating the p53 program for therapeutic purposes.